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In mice, CD49f hi  mammary stem cells (MaSCs) asymmetrically divide to generate CD49f +  committed progenitor cells that differentiate into CD49f −  phenotypes of the milk‐secreting tissue at the onset of pregnancy. We show CD49f +  primary mammary epithelial cells (PMECs) isolated from lactating tissue uniquely respond to pregnancy‐associated hormones (PAH) compared with CD49f +  cells from nonlactating tissue. Differentiation of CD49f +  PMEC in extracellular matrix produces CD49f −  luminal cells to form differentiated alveoli. The PAH prolactin and placental lactogen specifically stimulate division of CD49f −  luminal cells, while receptor activator of nuclear factor (NF)‐κB ligand (RANKL) specifically stimulates division of basal CD49f +  cells. In nondifferentiating conditions, we observed a greater proportion of multipotent self‐renewing cells, and RANKL treatment activated the RANK pathway in these cultures. Furthermore, we observed the deposition of calcium nodules in a proportion of these cells. These data imply that a MaSC unique to the lactating breast exists in humans, which generates progeny with discrete lineages and distinct response to PAH. S TEM  C ELLS  2012;30:1255–1264

Receptor Activator of NF‐κB Ligand Promotes Proliferation of a Putative Mammary Stem Cell Unique to the Lactating Epithelium